Scarab Genomics? C-Flow? technology is a highly efficient continuous culture system that can produce kilograms of protein in a few weeks at a mere 10-liter scale. This project will determine whether C-Flow can be further developed as an efficient system for production of single-chain antibody therapeutics. Antibody fragments, especially single-chain variants, are increasingly important for diagnostic and therapeutic use such as toxin and virus neutralization, being relatively easy to manufacture in E. coli, albeit very inefficiently in current techniques. They are of great potential significance as therapies for outbreaks and biothreats, with notable success against Ebola in West Africa. Other antibodies are remarkably successful for e.g. treatment of cancer and autoimmune diseases. These molecules have a variety of structures and indications but severe problems in manufacturing. Lacking glycosylation, antibody fragments are short-lived in vivo, requiring multiple high doses, leading to prohibitively expensive drugs. Therefore, a method of production that is fast, efficient, and low in cost is critically needed. This project will optimize C-Flow production of three structurally distinct single chain fragment antibodies, to evaluate the general applicability of the approach. The goal is high expression levels and sustained production on an unprecedented scale with small-footprint equipment. Correct folding of antibody fragments is critical for function. E. coli mechanisms for expression and delivery into the periplasm, where protein folding occurs, include chaperones, signal sequences, and codon usage and distribution. These mechanisms will be optimized for all three antibody fragments. Computer predictions have the power to reveal folding defects that could be corrected by genetic engineering to replace residues or regions that do not fold well. By testing an example immunotoxin that does not express strongly, the possibility of using software predictions for rational antibody design for increased manufacturability will be explored. Engineered changes suggested by computer predictions will be implemented and evaluated. Such an integrated system of software and production technology could provide a rapid response to a bio-emergency, going from outbreak to therapeutic ready for clinical testing in weeks. The Specific Aims are: 1. Optimize production of a structurally simple antibody fragment, scFv-SG1, at 1-liter then 10-liter C-Flow scales, extending the latter to at least 30 days to evaluate continuous antibody production. 2. Optimize production of a more structurally complex antibody fragment, scFabYMF10 as per Aim 1, purify the antibody and test its function by evaluating scFabYMF10 binding to its target antigen. 3. Enhance expression of the antibody-toxin conjugate B3Fv-PE40 (poorly expressed in E. coli) using physiological and genetic engineering approaches. Determine whether up-front software-assisted design of the protein can be used to predict improved manufacturability.